Gelatin Zymography Kit (Cosmo Bio type: narrow thin plates)

Cosmo Bio

Catalog No.: PMC-AK47

Product Description

Matrix metalloproteinases (MMPs) belong to the family of metalloproteinases, which consists of at least 20 members, and known to be involved in the metabolism of extracellular matrix proteins. MMPs are widely detected by zymography. The Gelatin-Zymography Kit provides an easy system of the electrophoresis for zymography. This product is used for detecting ProMMP-2, MMP-2 and ProMMP-9 in blood, body fluid, secretion, cell lysate, cell culture medium, and other samples.
Our Zymography precast gels are available in two types (sizes). Please choose the type that fits your gel box. 
     ATTO type: Glass plate gel is 120(W)×100(H) and the thickness is 1mm
     Cosmo Bio type: Glass plate gel is 100(W)×100(H) and the thickness is 1mm.  
Zymography Kit provides an easy system of the electrophoresis for zymography. This product is used for detecting ProMMP-2, MMP-2 and ProMMP-9 in blood, body fluid, secretion, cell lysate, cell culture medium, and other samples. [Cautions: This product has a short expiration date (less than 3 months). Please use it after arrival as soon as possible.

Protocol

1. Load 100-150 mL of Electrophoresis Buffer to the lower chamber (anode). Refer to the instruction manual ofyour electrophoresis tank/chamber to know appropriate volume of the buffer.
2. Take out the comb on precast gel carefully and set the gel to electrophoresis chamber. The side of sample holes should be set on the upper side. If the sample holes aredisturbed, fix them up with needle etc.
3. Load around 100 mL of Electrophoresis Buffer to the upper chamber (cathode).
4. Mixsamples with equivalent volume of sample preparation buffer. Incubate for 15 minutes at Room Temperature. (Do NOT heat thesamples.)
*MMP markers can be used without sample preparation buffer.
5. Apply the samples and MMP markers to gel plate.
6.Run electrophoresis at 15 mA constant current. (If you use 2 gels, set the current at 30 mA)
7. After the run is completed, turn off the electrophoresis chamber, and take out the gel plate from the chamber.
8. Remove the upper glass plate of thegel plate and peel off the gel carefully with a spatula.
9. Put the gel in the tray with 200 ml of Washing Buffer. Incubate with shaking at Room Temperature.
10. Put the gel in the container with 50 ml of Reaction Buffer and seal up the container. Incubate the gel in the container in incubator at 37°C for 20-40 hrs (Lower enzyme concentration needs longer reaction time.).
11. After enzymatic reaction, put the gel in the container with Staining solution. Incubate for 30 minutes at Room Temperature to stain protein.
12. Put the gel in the container with De-staining solution. And incubate for 30 minutes to several hours to de-stain.
Gelatin Zymography Kit provides an easy system of the electrophoresis for zymography. This product is used for detecting ProMMP-2, MMP-2 and ProMMP-9 in blood, body fluid, secretion, cell lysate, cell culture medium, and other samples. [Cautions: This product has a short expiration date (less than 3 months). Please use it after arrival as soon as possible.

•    Jong Youl Kim, Nuri Kim, Zhen Zheng, Jong Eun Lee, and Midori A. Yenari
The 70kD heat shock protein protects against experimental traumatic brain injury
Neurobiol Dis. 2013 October ; 58: 289–295. PMID: 23816752
•    Yang Zheng, Seiji Yamamoto, Yoko Ishii, Yang Sang, Takeru Hamashima, Nguyen Van De, Hirofumi Nishizono, Ran Inoue, Hisashi Mori, and Masakiyo Sasahara
Glioma-Derived Platelet-Derived Growth Factor-BB Recruits Oligodendrocyte Progenitor Cells via Platelet-Derived Growth Factor Receptor-a and Remodels Cancer Stroma
The American Journal of Pathology, Vol. 186, No. 5, May 2016 PMID: 26945107

References

•    Miyazaki, K., Ohta, Y., Nagai, M., Morimoto, N., Kurata, T., Takehisa, Y., Ikeda, Y., Matsuura, T., Abe, K.
Disruption of Neurovascular Unit Prior to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis.
J. Neurosci. Res. 89, 718-728 (2011)
PMID: 21337372
•    Fujiwara, M., Kashima, T. G., Kunita, A., Kii, I., Komura, D., Grigoriadis, A. E., Kudo, A., Aburatani, H., Fukayama, M.
Stable Knockdown of S100A4 Suppresses Cell Migration and Metastasis of Osteosarcoma.
Tumour Biol. 2011 Jun;32(3):611-22.
PMID: 21360024
•    Aoki, T., Kataoka, H., Ishibashi, R., Nozaki, K., Hashimoto, N.
Simvastatin Suppresses the Progression of Experimentally Induced Cerebral Aneurysms in Rats.
Stroke. 39, 1276-85 (2008) PMID: 18309148
•    Kuramochi, D., Unoki, H., Bujo, H., Kubota, Y., Jiang, M., Rikihisa, N., Udagawa, A., Yoshimoto, S., Ichinose, M.,Saito, Y.
Matrix Metalloproteinase 2 Improves The Transplanted Adipocyte Survival in Mice.
Eur. J. Clin. Invest. 38, 752-759 (2008) PMID: 18837800
•    Fujimoto, M., Takagi, Y., Aoki, T., Hayase, M., Marumo, T., Gomi, M., Nishimura, M., Kataoka, H., Hashimoto, N., Nozaki, K.
Tissue Inhibitor of Metalloproteinases Protect Blood-brain Barrier Disruption in Focal Cerebral Ischemia.
J. Cereb. Blood Flow Metab. 28, 1674-1685 (2008) PMID: 18560439

 

Citations for Gelatin Zymography Kit (Cosmo Bio type: narrow thin plates) – 11 Found

Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells. Scientific Reports. 2016;6( 27572741):32248. 

Omiya, Shigemiki; Omori, Yosuke; Taneike, Manabu; Protti, Andrea; Yamaguchi, Osamu; Akira, Shizuo; Shah, Ajay M; Nishida, Kazuhiko; Otsu, Kinya. Toll-like receptor 9 prevents cardiac rupture after myocardial infarction in mice independently of inflammation. American Journal Of Physiology. Heart And Circulatory Physiology. 2016;311(6):H1485-H1497. 

Egashira, Yusuke; Zhao, Hao; Hua, Ya; Keep, Richard F; Xi, Guohua. White Matter Injury After Subarachnoid Hemorrhage: Role of Blood-Brain Barrier Disruption and Matrix Metalloproteinase-9. Stroke. 2015;46(10):2909-15. 

Enomoto, Daichi; Obana, Masanori; Miyawaki, Akimitsu; Maeda, Makiko; Nakayama, Hiroyuki; Fujio, Yasushi. Cardiac-specific ablation of the STAT3 gene in the subacute phase of myocardial infarction exacerbated cardiac remodeling. American Journal Of Physiology. Heart And Circulatory Physiology. 2015;309(3):H471-80. 

Fumoto, Toshio; Naraoka, Masato; Katagai, Takeshi; Li, Yuchen; Shimamura, Norihito; Ohkuma, Hiroki. The Role of Oxidative Stress in Microvascular Disturbances after Experimental Subarachnoid Hemorrhage. Translational Stroke Research. 2019;10(6):684-694. 

Takagi, Yuya; Aoshima, Kota; Kuse, Yoshiki; Nakamura, Shinsuke; Okada, Yasunori; Shimazawa, Masamitsu. The Involvement of HYBID in the Regulation of Intraocular Pressure and Extracellular Matrix Accumulation in the Trabecular Meshwork. Biological & Pharmaceutical Bulletin. 48(10):1555-1565. 

Hoang, Thi Xoan; Dang, Nhat Minh; Bae, Kang Gyu; Kim, Jae Young. Anti-Inflammatory and Antioxidant Effects of Topical Formulations Containing Plant Extracts, Methylsulfonylmethane, and Peptiskin(®) in In Vitro Models of Arthritis. Pharmaceuticals (Basel, Switzerland). 2025;18(9) 

Ishida, Yuko; Kuninaka, Yumi; Komori, Tadasuke; Iwabuchi, Sadahiro; Nosaka, Mizuho; Kimura, Akihiko; Hashimoto, Shinichi; Morikawa, Yoshihiro; Mukaida, Naofumi; Kondo, Toshikazu. OSMRβ-mediated signals on resident fibroblasts restore healing in diabetic skin wounds through promoting angiogenesis and granulation tissue formation. Communications Biology. 2025;8(1):1505. 

Ogawa, Hisataka; Ekawa, Tomoya; Mizote, Yu; Akazawa, Takashi; Tahara, Hideaki. Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors. Frontiers In Immunology. 16:1506204. 

Choudhury, Mohammed E; Ozaki, Saya; Miyaue, Noriyuki; Matsuura, Taisei; Mikami, Kanta; Islam, Afsana; Kubo, Madoka; Ando, Rina; Yano, Hajime; Kunieda, Takeharu; Nagai, Masahiro; Tanaka, Junya. Chloride Intracellular Channel Protein 2 Promotes Microglial Invasion: A Link to Microgliosis in the Parkinson's Disease Brain. Brain Sciences. 2022;13(1) 

Shimada, Yoshiaki; Shimura, Hideki; Tanaka, Ryota; Yamashiro, Kazuo; Koike, Masato; Uchiyama, Yasuo; Urabe, Takao; Hattori, Nobutaka. Phosphorylated recombinant HSP27 protects the brain and attenuates blood-brain barrier disruption following stroke in mice receiving intravenous tissue-plasminogen activator. Plos One. 13(5):e0198039. 

Gelatin Zymography Kit (Cosmo Bio type: narrow thin plates) FAQs

What is zymography?
Zymography is a method for detecting enzymes based on their enzymatic activity after separation by electrophoresis. When a gel containing a substrate protein such as gelatin or casein is used, the entire gel is stained blue after protein staining. Regions with enzymatic activity appear as clear bands because the substrate protein has been degraded.

Why can proMMPs be detected as bands in zymography even though they are inactive enzymes? Why do proMMPs and MMPs have different molecular weights?
proMMPs are inactive because their active sites are masked by a propeptide sequence. During zymography, SDS treatment releases the propeptide sequence masking the active site, allowing even proMMPs to exhibit enzymatic activity. MMPs are activated when the propeptide region is cleaved from proMMPs. Therefore, precursor proMMPs have a higher molecular weight, whereas activated MMPs have a lower molecular weight.
For more information, please refer to the following publication:
Possible role of matrix metalloproteinases in reconstruction of peripheral pulmonary arteries induced by hypoxia. J. Novotná and J. Herget Physiol Res. 2002;51(4):323-334.

What is the gel concentration of the precast gel included in Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
The acrylamide concentration is 10%, and the gel contains 0.1% gelatin.

What are the dimensions of the precast gel included in Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
Gel size: 100 × 100 mm
Gel thickness: 1 mm
Overall thickness including glass plates: 3.6 mm

How long does electrophoresis take at 15 mA when using Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
Depending on the electrophoresis apparatus used, electrophoresis typically takes approximately 3 hours.

How can I obtain clear images of zymography gels?
For optimal visualization of bands, it is recommended to illuminate the gel from below using white light.

How much protein should be loaded per lane when using Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
Since MMP activity varies depending on the tissue or cell type, it is difficult to define a universally optimal amount of protein to load. As an initial guideline, load approximately 10 µg of protein and determine the optimal amount based on the results obtained. When culture supernatants are used as samples, the volume of the sample is often used as the loading criterion.

How can MMPs be extracted from tissues?
Since MMPs are tightly associated with extracellular matrix components in tissues, extraction efficiency is generally low.
One method is to homogenize tissue using a Polytron homogenizer in a buffer containing 10 mM CaCl₂ and 0.25% Triton X-100, followed by centrifugation. The resulting supernatant can then be used as the sample.
Other extraction methods include urea solubilization and heat treatment at 60°C. Additional information is available in Woessner Jr., Methods in Enzymology, Vol. 248, pp. 510-529.

What should be avoided during sample preparation for Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
The use of reducing agents such as 2-mercaptoethanol and dithiothreitol (DTT), as well as heat treatment, should be avoided because they may denature proteins and result in loss of enzymatic activity.

Is there any information regarding the effects of blood collection anticoagulants on samples used with Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
Gerlach et al. (Analytical Biochemistry, Vol. 344, pp. 147-149) reported a comparison of serum and plasma prepared using three anticoagulants: citrate, heparin, and EDTA. Please refer to the publication for details.

What is the band detected between proMMP2 and MMP2 when using Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
The band corresponds to intermediate MMP2. MMP2 is activated by cleavage of the propeptide region from proMMP2, and intermediate MMP2 represents a processing intermediate that appears during this activation process.

Is the mobility of proMMP9 different between mice and humans when using Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
Yes. MMP9 contains both N-linked and O-linked glycans, and the size of these glycans differs among animal species. Therefore, mouse-derived proMMP9 is detected at a higher molecular weight than human-derived proMMP9.

What could the bands above proMMP9 represent when using Gelatin Zymography Kit (Cat. No. PMC-AK47) and FITC-labeled Gelatin Zymography Kit (Cat. No. PMC-AK83)?
The bands may represent MMP9 dimers or complexes formed between MMP9 and other proteins, such as TIMPs, lipocalin, or α2-macroglobulin.